The dynamin GTPase mediates regenerative axonal fusion in Caenorhabditis elegans by regulating fusogen levels

Abstract Axonal fusion is a neuronal repair mechanism that results in the reconnection of severed axon fragments, leading to the restoration of cytoplasmic continuity and neuronal function. While synaptic vesicle recycling has been linked to axonal regeneration, its role in axonal fusion remains unknown. Dynamin proteins are large GTPases that hydrolyze lipid-binding membranes to carry out clathrin-mediated synaptic vesicle recycling. Here, we show that the Caenorhabditis elegans dynamin protein DYN-1 is a key component of the axonal fusion machinery. Animals carrying a temperature-sensitive allele of dyn-1(ky51) displayed wild-type levels of axonal fusion at the permissive temperature (15°C) but presented strongly reduced levels at the restrictive temperature (25°C). Furthermore, the average length of regrowth was significantly diminished in dyn-1(ky51) animals at the restrictive temperature. The expression of wild-type DYN-1 cell-autonomously into dyn-1(ky51) mutant animals rescued both the axonal fusion and regrowth defects. Furthermore, DYN-1 was not required prior to axonal injury, suggesting that it functions specifically after injury to control axonal fusion. Finally, using epistatic analyses and superresolution imaging, we demonstrate that DYN-1 regulates the levels of the fusogen protein EFF-1 post-injury to mediate axonal fusion. Together, these results establish DYN-1 as a novel regulator of axonal fusion.


Introduction
Regenerative axonal fusion promotes the functional recovery of injured neurons by reuniting severed segments of an axon (1)(2)(3)(4)(5). This process has been best characterized in the Caenorhabditis elegans (C. elegans) posterior lateral microtubule (PLM) axons, which undergo spontaneous regrowth, reconnection, and fusion that restores the structure and function of the neuron after injury (2)(3)(4)(5). Molecules previously implicated in the recognition of apoptotic cells have been shown to be repurposed in the nerve repair pathway to promote regrowth and reconnection after axonal injury. For example, immediately following cell death, the normally internalized phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane where it is bound by the PS receptor PSR-1 to mediate cell corpse recognition (6). Meanwhile, the ABC transporter protein CED-7 and transthyretin protein TTR-52 facilitate the release of PS-exposing vesicles from the dying cell to the extracellular space to further attract engulfing cells (7). Simultaneously, the intracellular adaptor CED-1/LRP1 binds to TTR-52 and the lipidbinding protein NRF-5 at the plasma membrane and initiates a signaling cascade via the CED-1 adaptor protein CED-6/GULP (7)(8)(9). During the axonal fusion process, all of these proteins except CED-1 function to enable regrowth and recognition between severed axon fragments (3). Although the GTPase protein DYN-1/ dynamin also functions in the recognition of dying cells (10), its role in axonal repair has not previously been reported.
The dynamin GTPases are large proteins that mediate vesicle scission during endocytosis and synaptic vesicle recycling. Pivotal studies conducted in Drosophila melanogaster reported the participation of Shibire/dynamin 1 in neurotransmission and synaptic vesicle recycling (11)(12)(13)(14). Other proteins linked to neurotransmission have previously been reported to participate in the regeneration of the C. elegans PLM axons, including the microtubule-associated motor protein UNC-57/endophilin (15). Since the mammalian dynamin proteins are known to associate with microtubules (16) and function in the apoptotic recognition pathway (10,17), we hypothesized that the dynamin GTPases may also have a role during regenerative axonal fusion. Here, we confirm this hypothesis by establishing that the DYN-1 protein is important for facilitating axonal fusion and regrowth after injury.
In C. elegans, axonal fusion is reliant on the transmembrane glycoprotein epithelial fusion failure-1 (EFF-1), which is structurally homologous to class II viral fusion proteins (18,19). Animals lacking EFF-1 display extensive branching and physical connection between severed axon segments but fail to fuse these segments (3,20). Further investigations using the C. elegans bilateral pair of PLM neurons revealed that EFF-1 mediates homotypic fusion between PLM axon segments in a manner resembling that in hypodermal and muscle cells (3,(20)(21)(22)(23)(24). In addition, the metalloprotease ADM-4 stabilizes EFF-1 to allow the membranes to merge (25). A recent study also reported that the small GTPase RAB-5 facilitates the endosomal recycling of EFF-1 in the PLM soma and increases the capacity of the neuron to undergo regenerative axonal fusion (26). Other interactors of EFF-1 have been identified in the context of cell-cell fusion, epidermal wound healing, and neuronal maintenance (24,(27)(28)(29)(30). However, how EFF-1 is regulated in the axon where it is required to drive the fusion of separated axon segments has not previously been reported.
Here, we demonstrate that DYN-1 controls the levels of EFF-1 to promote axonal fusion.

DYN-1 is important for axonal fusion
To analyze the role of DYN-1 in axonal fusion, we used the only viable mutant allele (ky51), which introduces a P70S point mutation that results in a temperature-sensitive loss of DYN-1 function: the DYN-1 protein degrades at restrictive temperatures (25°C) but functions normally at permissive temperatures (15°C) (31,32). The P70S allele affects the GTPase domain, which is required to hydrolyze the lipid membrane and facilitate the oligomerization of DYN-1 proteins at the site of scission (31,32). To study regenerative axonal fusion, the C. elegans PLM axon was severed ∼50 µm from the cell body with a Micropoint UV laser system (Fig. 1A). Successful fusion events were recorded 48 h after injury only when membrane integrity and cytoplasmic continuity were established between the two fragments ( Fig. 1B) (3)(4)(5). At the restrictive temperature, dyn-1(ky51) animals displayed a strong reduction in the level of axonal fusion (Fig. 1C). However, when grown at 15°C (permissive for DYN-1 function), the level of fusion in dyn-1(ky51) animals was indistinguishable from wild-type (WT) controls (Fig. 1C). We further analyzed regeneration by recording the length of regrowth in axons that did not establish physical contact with their severed segments (Fig. 1D). The length of axonal regrowth 24 h after ablation was significantly reduced in dyn-1 animals grown at the restrictive temperature (Fig. 1E). Thus, DYN-1 is involved in both axonal fusion and regrowth pathways.
To understand the temporal regulation of DYN-1 during axonal fusion, we grew dyn-1(ky51) animals at either 15°C (permissive temperature) or 25°C (restrictive temperature) prior to axotomy before either keeping them at the same conditions or shifting them to the alternative temperature after axotomy. As shown in Fig. 1F, dyn-1(ky51) animals displayed significantly reduced axonal fusion when incubated at 15°C pre-axotomy and transferred to 25°C immediately afterward. In contrast, animals incubated at 25°C pre-axotomy and transferred at 15°C post-axotomy displayed no defect in axonal fusion (Fig. 1F). Thus, these experiments suggest that DYN-1 is important after injury for axonal fusion. Furthermore, additional temperature-shift experiments indicate that DYN-1 functions throughout the regenerative process to promote axonal fusion ( Fig. S1A and B).

DYN-1 likely functions cell-autonomously to facilitate regrowth and repair
To confirm the involvement of DYN-1 in axonal regrowth and fusion, we performed genetic rescue experiments by expressing WT dyn-1 specifically in the six mechanosensory neurons (including the two PLM neurons) of dyn-1(ky51) animals. Transgenic strains harboring extrachromosomal arrays of WT dyn-1 displayed partial rescue of the deficiencies in axonal fusion and regrowth lengths (Fig. S1C). To further confirm the role of DYN-1 in these phenotypes, we used CRISPR-Cas9 to introduce either of the two dynamin isoforms (1a and 1b) into dyn-1(ky51) animals ( Fig. 2A). Isoform 1a partially, but significantly, rescued the fusion defect and fully rescued the regrowth length defect (Fig. 2B). Interestingly, isoform 1b failed to rescue either defect, suggesting that only isoform 1a is involved in axonal repair ( Fig. 2B and C; bars [3][4][5][6]. This rescue data demonstrate that DYN-1 likely functions cell-autonomously to mediate axonal fusion and regrowth. We next expressed dyn-1 from its endogenous promoter in dyn-1(ky51) animals to determine whether this could further enhance the levels of axonal fusion and the length of regrowth. Interestingly, this transgene only partially rescued the axonal fusion and fully rescued the regrowth length defects in dyn-1 animals to similar levels as those seen with the cell-autonomous rescue transgenes ( Fig. 2B and C; bars 7 and 8). Together, these data suggest that DYN-1 may act cell-autonomously in the PLM neuron to promote axonal repair.

DYN-1 interacts with EFF-1 to promote axonal fusion
The nematode-specific fusogen EFF-1 is a key molecule for the process of axonal fusion (3) and is known to interact with DYN-1 to regulate hypodermal cell fusion (29). Hence, we examined the genetic interaction between eff-1 and dyn-1 in the context of axonal fusion. Axonal fusion levels were assessed in eff-1(ok1021) and dyn-1(ky51) single-and double-mutant animals at the restrictive temperature (25°C). Both dyn-1 and eff-1 single mutants displayed strong reductions in fusion levels compared with the wild type (Fig. 3A). Importantly, eff-1; dyn-1 double-mutant animals also displayed significant reductions in fusion levels that were not worse than either of the single-mutant strains (Fig. 3A), indicating that dyn-1 and eff-1 function in the same genetic pathway. Loss of EFF-1 did not affect the average length of regrowth and did not worsen the level observed in dyn-1(ky51) mutants (Fig. S2A). Thus, dyn-1 and eff-1 appear to function in the same genetic pathway during axonal fusion, with EFF-1 not required for axonal regrowth.
To further examine the interaction between EFF-1 and DYN-1, we overexpressed EFF-1 in animals carrying the dyn-1(ky51) mutation. To overexpress EFF-1, we used the vdEx662 transgene, which contains an mCherry fluorophore driven by a PLM-specific promoter (mec-4) and a GFP-tagged version of EFF-1 expressed under the same promoter (3). After injury, EFF-1 localizes to the membrane to fuse the separated segments back together (3) (Fig. S2B-D). We performed UV laser axotomy in EFF-1 overexpressing animals carrying the dyn-1(ky51) mutation and observed WT levels of fusion, suggesting that EFF-1 lies downstream to DYN-1 (Fig. 3B).

DYN-1 is important for the expression of EFF-1 immediately after injury
Using superresolution microscopy and a UV laser system, we measured the levels of EFF-1 at different time points after injury. Interestingly, we found that EFF-1 levels were significantly reduced in dyn-1(ky51) animals on both proximal and distal axon segments at the earliest time points following injury (measured at 15  WT and dyn-1 animals at permissive and restrictive temperatures. dyn-1 animals display a significant defect at 25°C but not at 15°C, compared with WT animals. P-values calculated using the unpaired t test where ***P < 0.001. F) Axonal fusion levels in WT and dyn-1 animals were grown at 15°C before and after axotomy (bars 1 and 2), at 15°C before axotomy and at 25°C afterward (bars 3 and 4), at 25°C before axotomy and at 15°C after axotomy (bars 5 and 6), and at 25°C before axotomy followed by 25°C post-injury (bars 7 and 8). Error bars represent standard error of proportion; P-values calculated using Fisher's exact tests; *P < 0.05 and **P < 0.005. Numbers within individual bars indicate the number of reconnection events per genotype, where n ≥ 15 reconnection events.
levels were unchanged between dyn-1(ky51) and WT animals 24 h postaxotomy, and the loss of dyn-1 did not affect the localization of EFF-1 to the membrane post-axotomy (Fig. S2). Overall, these data imply that DYN-1 is essential for the correct regulation of EFF-1 levels soon after injury, but not so at later time points in the proximal segment.
To address whether DYN-1 can affect the expression of eff-1, we used a Peff-1::GFP transgene and confocal microscopy to visualize the levels of GFP expressed from the eff-1 promoter. We quantified the corrected total cell fluorescence (CTCF) intensity of GFP in the PLM cell body prior to axotomy and 1 h after severing the PLM axon. While WT animals displayed an increase in expression from the eff-1 promoter, those lacking DYN-1 displayed a slight reduction in expression compared with the levels before axotomy levels ( Fig. 5A-C). Thus, DYN-1 is important for the correct expression and levels of EFF-1 after axotomy.
Together, these data suggest that DYN-1 mediates axonal fusion by regulating the levels of EFF-1, which is required to fuse the two membranes once reconnection between the proximal and distal segments occurs.

Characterizing DYN-1 in axonal repair
In this study, we have elucidated a novel role for the DYN-1/dynamin GTPase in axonal fusion. Axonal fusion levels in animals carrying the dyn-1(ky51) allele were higher at the permissive temperature and significantly lower at restrictive temperatures. Importantly, this defect was observed only after axotomy, suggesting that DYN-1 is not necessarily required for the development of the neuron but for its maintenance. This is in line with previously published reports in mice where individual gene knockouts of dynamin isoforms did not affect the development of a large nerve terminal (33). We further report that the DYN-1 protein is important for several hours post-injury to maintain regenerative axonal fusion. Our previous analysis of microtubule dynamics before and after axotomy provides further support for this notion (34). In the absence of axonal injury, dyn-1 mutants displayed no noticeable defect in newly forming microtubules or in the polarity of microtubule growth (34). Similar observations were made by Noda et al. (35) in intact mammalian neurons, who suggested that dynamin participates in vesicular trafficking rather than microtubule sliding as a motor protein. However, our post-axotomy data suggest that DYN-1 might be involved in microtubule production, bundling, and polarity soon after injury (34). Thus, DYN-1 might regulate axonal regrowth and fusion by controlling microtubule dynamics and maintaining the polarity of the microtubule network. Indeed, microtubule regulation has previously been implicated in PLM axon regeneration wherein loss of function of microtubulebinding proteins has affected axonal repair after injury (15,36,37).
We propose that dyn-1 might temporally regulate axonal regrowth and fusion depending on its availability in the cell. Both axonal fusion and regrowth lengths were rescued when WT dyn-1 was reintroduced cell-autonomously. However, the overexpression of DYN-1 induced only axonal fusion defects, with regrowth length remaining unaffected. In a study conducted in Shibire/dynamin temperature-sensitive mutants in D. melanogaster, overexpression of dynamin resulted in poor neurotransmission, vacuolated mitochondria, and cross-linked microtubules (38). Since dynamin GTPases largely depend on the availability of GTP in the cell, it is likely that the imbalance of enzyme and substrate causes a delay in endosomal trafficking, thereby phenocopying the loss of gene function. It is also possible that the overexpression of dynamin in the neuron favors apoptosis or the "eat-me" signaling pathway as seen in metastatic cancer cells (39). These results may also suggest that regrowth and axonal fusion are regulated by two separate pathways, supporting previously published findings (4).

Dynamin and EFF-1
This study sheds light on the interaction between DYN-1 and EFF-1 and the possibility that DYN-1 functions in axonal repair through two distinct pathways. EFF-1 and DYN-1 were previously implicated in hypodermal cell-cell fusion wherein DYN-1 was required for the uptake of EFF-1 from the plasma membrane to allow its recycling (29). In our data set, we observed a similar trend in EFF-1 expression in the distal fragments of axons that failed to reconnect. The expression of EFF-1 in the proximal fragment in dyn-1 mutant animals displayed the largest difference to control axons soon after axotomy. This disruption in the accumulation of EFF-1 proximal to injury might be a direct consequence of disoriented microtubules post-injury (34), suggesting that DYN-1 may influence the transport of growth-promoting molecules by bundling microtubules. As the overexpression of EFF-1 resulted in a complete rescue of axonal fusion defects in dyn-1 mutant animals, it suggests that EFF-1 lies downstream to DYN-1 in the fusion pathway. Thus, there are at least three possibilities arising from our experimental data. First, DYN-1 is required in the proximal fragment transiently after injury to transport vesicles. Second, DYN-1 recycles EFF-1 vesicles in the distal fragment over the first several hours after injury. Third, DYN-1 influences the availability of functional EFF-1 in the cell body after axonal injury. The RAB-5 GTPase has previously been implicated in recycling EFF-1 in C. elegans embryos and in the PLM axon (29,31). Thus, in the absence of DYN-1, the RAB-5/Rab5 GTPase likely transports and recycles EFF-1. While DYN-1 is not involved in this uptake of EFF-1 in intact PLM axons (31), it is not known whether DYN-1 is required for the endocytosis or transport of EFF-1 after injury. DYN-1 may also be required for the endocytosis of molecules in the apoptotic recognition machinery that are known to be repurposed to facilitate axonal fusion (3). Together, our data indicate that DYN-1 is a major player in axonal repair and likely elicits injury responses through multiple pathways.

C. elegans maintenance
Animals were maintained on nematode growth media (NGM) at 15°C unless otherwise specified, using standard methods (40). Some strains were obtained from the Caenorhabditis Genetics Center (CGC), Minnesota, USA. The dyn-1(ky51); zdIs5 strain was generated using standard genetic crossing. The CRISPR-generated strains sybIs2534 and sybIs2535 were designed by us and generated by SunyBiotech (Fujian Province, China). Varied concentrations (1 ng/µL, 5 ng/µL, and 10 ng/µL) of PLM-specific dynamin rescue and 5 ng/µL of dynamin promoter expressing genomic DNA with a C-terminal fluorescent tag (WrmScarlet) were generated using standard techniques (41). All double mutants were generated using standard genetic crossing techniques. A full list of strains used in this study is shown in Table S1.

UV laser axotomy
PLM axons were severed with a Micropoint UV laser system (Andor -Oxford Instruments, Belfast, UK) attached to Zeiss Axio Imager.A2 compound microscope (Zeiss Group, Oberkochen, Germany), ∼50 µm from the cell body (2,3). The levels of reconnection and fusion were recorded 24 and 48 h after axotomy, respectively, using a Zeiss Axio Imager.M2 microscope (Zeiss Group, Oberkochen, Germany). The length of regrowth from the proximal fragment was measured at the 24-h time point from the cut site to the distal most point. For animals that were incubated at 25°C before axotomy, gravid adults were bleached and washed to obtain synchronized eggs and incubated at 15°C for 48 h until they were viable L2 stage worms. Midlate L4 animals were obtained when L2 animals were incubated at 25°C for 16 h.
Precast 4-15% acrylamide gels (Bio-Rad) were used for SDS-PAGE, and proteins were transferred to polyvinylidene difluoride (PVDF) membranes (100 V for 2 h). Membranes were blocked using TBS with 5% BSA and 0.1% Tween 20. They were then washed and incubated overnight at 4°C with primary antibodies: anti-eGFP (1:1,000) [Roche; from mouse immunoglobulin G1κ (clones 7.1 and 13.1) #11814460001] or anti-FLAG (1:1,000) (Sigma F1804). After washing, membranes were subsequently incubated for 1 h at room temperature with horseradish peroxidaseconjugated secondary antibodies (1:10,000). They were then washed extensively using TBS-T, prior to be analyzed with enhanced chemiluminescence methods and images being captured on an Odyssey Fc imaging system (LI-COR) with Image Studio Lite (LI-COR) software.

Localization of EFF-1::GFP
To examine EFF-1 expression levels in regrowing axons, animals were first incubated at restrictive temperature for at least 2 h prior to imaging. Laser axotomies were performed in animals carrying the vdEx662 transgene in 0.05% tetramisole in M9 and 4% agar pads. Bidirectional confocal imaging for pre-and postaxotomy samples was conducted for both WT and mutant animals on the same agar pad using 11% laser power for 488 nm and 0.6% for 561 nm. Animals were imaged before axotomy and at 15 min, 1 h, 4 h, 8 h, and 24 h after injury as stated in Neumann et al. (3), using the Airyscan Multiplex (MPLX)-Super Resolution (SR) 8Y mode, and representative images were taken using the Airyscan MPLX-SR-4Y mode on the Zeiss LSM 980 compound microscope (Zeiss Group, Oberkochen, Germany) and Zen 2 software.
To examine EFF-1 expression levels in the cell body, animals were first incubated at 25°C for at least 2 h prior to imaging. Animals were immobilized with 0.05% tetramisole in M9 on 4% agar pads. Bidirectional confocal imaging for pre-and 1 h postaxotomy samples was conducted for both WT and mutant animals on the same agar pad using 11% laser power for 488 nm and 7.5% for 561 nm. Animals were imaged using the Airyscan Multiplex (MPLX)-Super Resolution (SR) 8Y mode on the Zeiss LSM 980 compound microscope (Zeiss Group, Oberkochen, Germany) and Zen 2 software.
To measure transcriptional activity, the cell body was traced and the mean fluorescence, area, and integrated density were recorded using ImageJ software (National Institutes of Health, Bethesda, MD, USA). We then consistently traced an area of 58.169 µm 2 (length × width = 8.52 µm × 8.52 µm) outside the worm body in all images to measure the background signal. Due to the complexity of the experiment, we examined at least nine animals per genotype to measure the CTCF. The CTCF per sample was calculated using the following formula: CTCF = integrated density − (area of the traced cell body × mean fluorescence of the background signal).